Selection of Antibody Fragments by Means of the Filter-Sandwich Colony Screening Assay

Rauth, Sabine; Schlapschy, Martin; Skerra, Arne

doi:10.1007/978-3-642-01144-3_17

Cited by 4 publications

(10 citation statements)

References 16 publications

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“…This uncertainty in the IPTG concentration in the filter-sandwich assay also causes a problem in the induction timing. For appropriate induction, the colony size is a critical factor [14,44]; however, the colony continues to grow during the assay. Hence, the timing of the expression induction is crucial for proper expression.…”

Section: Discussionmentioning

confidence: 99%

“…Toxicity to E. coli can increase the background, resulting in several false-positive clones being obtained. This situation renders panning extremely difficult; it is not easy to establish single positive clones only through several rounds of panning [14]. Although the phage display is a powerful tool for establishing monoclonal antibodies, it is used less frequently than expected [39].…”

Section: Screening With a Phage Displaymentioning

confidence: 99%

“…The presence of antibody fragments bound to the membrane is then detected, and the spot is superimposed on the colony. This method circumvents the difficult technique of lifting the colony [14,36]. In addition, the filter-sandwich assay was further optimized.…”

Section: Filter-sandwich Assaymentioning

confidence: 99%

“…These antibody gene libraries are incorporated into a phagemid or plasmid and expressed in phage or Escherichia coli (E. coli). Further, panning [13] or colony assays [14] are performed to isolate scFvs possessing affinity to the antigen, thereby establishing monoclonal antibodies. This step, the screening of antibody libraries, is critical for establishing monoclonal antibody fragments with a high affinity and specificity against the antigen.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Colony Assay for Antibody Library Screening: Outlook and Comparison to Display Screening

Kato¹,

Hanyu²

2018

Antibody Engineering

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Recombinant monoclonal antibodies are established by screening the antibody libraries. To obtain antibodies with a high specificity and affinity, an efficient screening process with a highly diverse library including low background signals is necessary. One of the most extensively used methods is the phage display method. Although phage display screening is a powerful tool for enriching clones from vast libraries, it is not easy to identify single clones with an antigen recognition function only through several rounds of biopanning. The application of colony assays for screening antibody libraries can identify clones with a high reliability by a direct observation of the antibody-antigen binding during the screening process; however, the size of the library that can be dealt with is limited. This chapter describes the colony assay as a current screening technology used in recombinant monoclonal antibody production, the possible problems in this method, and discusses the outlook for this technology.

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Section: Discussionmentioning

confidence: 99%

Section: Screening With a Phage Displaymentioning

confidence: 99%

Section: Filter-sandwich Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Colony Assay for Antibody Library Screening: Outlook and Comparison to Display Screening

Kato¹,

Hanyu²

2018

Antibody Engineering

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“…Once colonies form, the filter is transferred to an antigen-coated membrane placed on an agar plate containing isopropyl -D-1-thiogalactopyranoside (IPTG) to induce scFv production. The scFv produced by the colonies diffuses out, binds to the antigen on the membrane, and its presence is detected by superimposing the spot on the colony (filter-sandwich assay) [16,17]. A gene encoding the scFv with affinity for the antigen is obtained.…”

mentioning

confidence: 99%

Single-step Colony Assay with Autoinduction of scFv Expression for the Screening of Antibody Libraries

Kato¹,

Hanyu

2019

BioTechniques

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We present a simple colony assay method for screening antibody libraries based on autoinduction of antibody fragment expression. This protocol eliminates the need for colony size monitoring and a separate induction step for single-chain Fv (scFv) antibody fragment expression. Here, scFvs are expressed in an automatic and timely fashion during the assay, resulting in high yields of positive clones and substantial time savings. The method was used successfully to establish monoclonal scFvs with high affinity and specificity against human IgG.

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Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins

Hanyu

Kato²

2020

Molecules

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Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and—most importantly—it enables high-throughput procedures.

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Selection of Antibody Fragments by Means of the Filter-Sandwich Colony Screening Assay

Cited by 4 publications

References 16 publications

Colony Assay for Antibody Library Screening: Outlook and Comparison to Display Screening

Colony Assay for Antibody Library Screening: Outlook and Comparison to Display Screening

Single-step Colony Assay with Autoinduction of scFv Expression for the Screening of Antibody Libraries

Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins

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